N. M. Apóstolo et al., In vitro an exBONPLA vitro germinationNDIA 25(1): of 5-15. 2016

ISSN: 0524-0476

IN VITRO AND EX VITRO GERMINATION OF THREE HANDROANTHUS ()

NANCY M. APÓSTOLO1, EZEQUIEL E. LARRABURU1, MIRIAM N. GIL2, MARÍA A. ZAPATER2 & BERTA E. LLORENTE1

Summary: Apóstolo, N. M., E. E. Larraburu, M. N. Gil, M. A. Zapater & B. E. Llorente. 2016. In vitro and ex vitro germination of three Handroanthus species (Bignoniaceae). Bonplandia 25(1): 5-15.

Handroanthus impetiginosus, H. ochraceus and H. lapacho, “lapachos”, are found in the Northwest of Argentina and have germination and conservation problems in their natural habitats. The germination of seeds under controlled conditions is an alternative that ensures the propagation. In this integrated study, in vitro and ex vitro germination, seed characteristics and seedling morphology of three species were analyzed. The length and width of the seeds, the width of the wings, the width and length of the seed body and embryo were measured. The germination capacity of the three species was determined during 12 months after seed harvesting, carrying out ex vitro and in vitro germination trials. The seeds and embryos of H. impetiginosus were significantly larger than those of other species. Handroanthus impetiginosus seeds maintained a high germination capacity over the 12 months (95%). Seed germination capacity of H. ochraceus gradually decreased four months after seed collection (63 to 47%), whereas that of H. lapacho decreased abruptly after eight months. No significant differences were observed between ex vitro and in vitro methods in H. impetiginosus and H. ochraceus. In the three species, the germination capacity is directly proportional to the size of seeds and seedlings.

Key words: Handroanthus impetiginosus, H. lapacho, H. ochraceus, seed, seedling.

Resumen: Apóstolo, N. M., E. E. Larraburu, M. N. Gil, M. A. Zapater & B. E. Llorente. 2016. Germinación in vitro and ex vitro de tres especies de Handroanthus (Bignoniaceae). Bonplandia 25(1): 5-15.

Handroanthus impetiginosus, H.ochraceus and H. lapacho, "lapachos”, se distribuyen en el NO Argentino y presentan inconvenientes de germinación y conservación en su ambiente natural. La germinación de semillas bajo condiciones controladas es una alternativa para asegurar la propagación de especies con este tipo de problemáticas. En el presente estudio integral, se analizó la germinación in vitro y ex vitro, las características de las semillas y la morfología de las plántulas de las tres especies de Handroanthus mencionadas. Para ello, se midió el largo y ancho de las semillas, el ancho de las alas de la cubierta seminal, el ancho y largo del cuerpo seminal y del embrión. El poder germinativo de las tres especies fue determinado durante 12 meses luego de la cosecha de las semillas. Fueron determinados los parámetros de las plántulas obtenidas in vitro y ex vitro. El tamaño de la semilla y embrión de H. impetiginosus

1 Laboratorio de Cultivo de Tejidos Vegetales (CULTEV), Dep. de Ciencias Básicas, Universidad Nacional de Luján, C.C. 221. Luján (6700), Buenos Aires, Argentina. E-mail: [email protected] 2 Facultad de Ciencias Naturales, Universidad Nacional de Salta, Av. Bolivia 5150, Salta (4400), Argentina.

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fue significativamente mayor que el de las otras especies estudiadas.H. impetiginosus mantuvo altos porcentajes de germinación hasta los 12 meses (95 %). H. ochraceus perdió gradualmente su poder germinativo a partir de los 4 meses posteriores a la cosecha de las semillas (63 a 47 %). Mientras que, en H. lapacho disminuyó abruptamente a los 8 meses luego de la cosecha. No se observaron diferencias significativas entre ambos tratamientos de germinación (in vitro y ex vitro) para H. impetiginosus y H. ochraceus. Las tres especies mostraron una relación directa entre la capacidad germinativa y las dimensiones de las semillas y características de las plántulas.

Palabras clave: Handroanthus impetiginosus, H. lapacho, H. ochraceus, plántula, semilla.

Introduction Handroanthus lapacho is a tree up to 20 m in height, with yellow flowers, and The Handroanthus belongs to the its distribution is restricted to a narrow Bignoniaceae and arose from the altitudinal belt (1400-1750 m) in the north segregation of the genus Gomes of Argentina (Jujuy and Salta). Even though ex DC. by molecular phylogenetic studies it is naturally protected, it is considered as (Grose & Olmstead, 2007a, b). Currently, the vulnerable by the International Union for species included in Tabebuia (Gentry, 1972) Conservation of Nature (IUCN) as it occurs are distributed in three genera: Roseodendron in a geographically fragmented area and Miranda, Handroanthus Mattos and Tabebuia its occurrence and quality of habitat are Gomes ex DC. In Argentina, there are only two declining. It is cultivated sporadically in species in Tabebuia: T. aurea and T. nodosa, and urban areas in Salta and Jujuy (IUCN, 2004; none in Roseodendron. Most of the native species Zapater et al., 2009). belong to Handroanthus: H. impetiginosus The seeds of Handroanthus are thin, (Mart. ex DC) Mattos, H. lapacho (K. Schum.) exalbuminous and have a winged seed S. Grose and H. ochraceus (Cham.) Mattos are coat. The seed body is cordiform, flat and distributed in northwest Argentina, whereas H. wide (Silva et al., 2004; Ferreira & Cunha, albus (Cham.) Mattos, H. pulcherrimus (Sandw.) 2000; Zapater et al., 2009). Costa et al. S. Grose and H. heptaphyllus (Vell.) Mattos are (2004) and Sampaio et al. (2007) have found in the northeast (Grose & Olmstead, studied the ontogeny of H. ochraceus seed. 2007b; Lozano & Zapater, 2008; Zapater et al., Morphological and physiological aspects 2009; Zuloaga et al., 2011). of the germination of H. impetiginosus Handroanthus impetiginosus is a tree 20-30 have been reported by Silva et al. (2004). m in height, with pink flowers, and it is found Morphological studies of the , seed and up to 1,300 m in the Tucumano-Oranense seedlings have been reported by different forest in the provinces of Jujuy, Salta, Tucumán authors (Corner, 1976; Carvahlo et al., 2008; and Catamarca, Argentina. It has the widest Ferreira & Cunha 2000; Souza et al., 2005). distribution in Argentina of the species in this Even though most species of Handroanthus genus and it is cultivated as an ornamental produce large quantities of seed, many of from northwest Argentina to the province of them have germination and conservation Buenos Aires. However, its natural area has problems in their natural habitats (Bocchese been reduced by anthropogenic activities (Silva et al., 2008; Carvahlo et al., 2008; Justiniano et al., 2004; Lozano & Zapater, 2008; Zapater et et al., 2000; Nery et al., 2008). In vitro al., 2009). germination has been little studied (Nery Handroanthus ochraceus is a tree of 30-35 et al., 2008), however some studies have m in height, with yellow flowers, and its range been carried out on the ex situ germination of distribution covers Salta and Jujuy provinces of several species of this genus (Silva et in Argentina (from 300 to 1200 m). It suffers al., 2004; Ferreira & Cunha, 2000; Nery from irregular reproduction linked to unseasonal et al., 2008). In vitro cultivation of seeds frosts (Zapater et al., 2009). is an appropriate method that allows the

6 N. M. Apóstolo et al., In vitro an ex vitro germination of Handroanthus

propagation of species with recalcitrant dishes were previously disinfected with seed, with preservation difficulties or that sodium hypochlorite (NaClO) (55 g.L-1 have problems with asexual reproduction. active chlorine) at 50% for 5 min and rinsed After in vitro germination the seedlings with sterilized distilled water. The paper was obtained can be acclimatized directly under sterilized in an autoclave at 1 atm (120 ºC) for greenhouse conditions or used as explants in 20 min. Three repetitions were carried out for vitro morphogenesis (Blakesley et al., 1996; each species at 0, 4, 8 and 12 months since Fay, 1994; Larraburu et al., 2012; Thorpe the seed collection. The seeds were stored in et al., 1991). Therefore this technique is the fridge at 5ºC between these periods. The appropriate for ex situ conservation plans germination dishes were kept in the culture for rare or threatened species and also for chamber at 25 ± 2ºC under continuous light. massive propagation of species of economic The germination percentage was weekly interest (Silva et al., 2005; Fay, 1994). evaluated during 30 days (germination In this study the in vitro and ex vitro capacity). The germination energy was germination of three species of Handroanthus determined at five days after the beginning that occur in northwest Argentina (H. of the each test of germination. impetiginosus, H. lapacho and H. ochraceus) were evaluated. The morphology of the seed Seed culture and seedlings was also analyzed so that The seeds were stored at 5ºC for a year their characteristics could be related to the after their collection. They were disinfected germination rate in the three species. after the removal of the wing portion of the seed coat. The disinfection was done by washing with tap water for 1 h, immersion in Materials and Methods 20% NaClO for 30 min with agitation and, finally, three rinses with sterilized distilled material water. Seeds of H. impetiginosus, H. lapacho and Ex vitro culture. The disinfected seed H. ochraceus (Bignoniaceae) were collected was grown in seedling trays previously from different habitats in the province of sterilized with 20% NaClO. The substrate Salta, Argentina. The seed samples were used was soil:peat:perlite (1:1:1), which was stored in paper envelopes after being left autoclaved at 120ºC (1 atm) for 45 min. to dry and then were kept in a fridge at 5ºC In vitro culture. For the in vitro germination until their analysis. The seed used for data of each species the disinfected seeds were on morphology, germination energy and grown in basic Woody Plant Medium (WPM) capacity were collected in the first season, (Lloyd and McCown 1980) suplemented whereas seed used for in vitro and ex vitro with 5 mM bencylaminopurine (BA), 5 g.L-1 culture trials were collected in the second activated carbón, 100 mg.L-1 myo-inositol, season. 20 g.L-1 sucrose and 6 g L-1 agar, at pH 5.8. The culture medium (15 ml) was divided Seed morphology into flat bottomed tubes 503 cm tubes and The parameters of 30 seeds of each species sterilized in an autoclave at 1 atm for 20 min. were recorded at the time of collection: Both the ex vitro and the in vitro cultures length, width and thickness of the seed, were maintained in a controlled chamber at width of the wings, width and length of the 24± 2° C, with a photoperiod of 16 h light seed body and width, length, thickness and and 25-26 mmol m-2s-1 relative humidity. midsection of the embryo (Fig.1G, H). After 30 days from the start of the cultures, the germination percentage was recorded Germination energy and capacity of the seed as well as the morphological parameters For each species, 20 to 30 seeds, of the seedlings obtained (lengths of the depending on their size, were germinated epicotyl and hypocotyl and the width of the on damp paper in Petri dishes. Seeds and cotyledons).

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Fig. 1. Seed of Handroanthus species. A: General aspect of the seed of H. impetiginosus. B: Embryo of H. impetiginosus. C: General aspect of the seed of H. ochraceus. D: Embryo of H. ochraceus. E: General aspect of the seed of H. lapacho. F: Embryo of H. lapacho. G-H: Parameters measured in the seeds and embryo of the species: I, width of the seed; II, length of the seed; III, width of the wings of the seed; IV, length of the seed body; V, width of the seed body; VI, width of the embryo; VII, length of the embryo. Bars: A-F: 1 cm.

Statistical analysis were the species (with three levels: H. lapacho, The experimental design was totally H. impetiginosus, H. ochraceus) and culture randomized in all the trials. Unifactorial method (with two levels: in vitro, ex vitro). analysis was applied for the seed characteristics. The data was analyzed with ANOVA and a Bifactorial analysis was used for the germination multiple mean comparison (Tukey Test, p< 0.05) percentage, the epicotyl and hypocotyl lengths using SPSS® 12.0 (SPSS Inc. Chicago, Illinois, and the cotyledon width. The factors considered USA).

8 N. M. Apóstolo et al., In vitro an ex vitro germination of Handroanthus

Results with cytokinins allowed the elongation of the stem of the seedling in all three species studied (Fig. 4 The seeds of the studied species showed A-C, F). morphological characteristics of the Bignoniaceae The ex vitro germination substrate family: winged seed coat, cordiform seed body (peat:perlite:soil, 1:1:1) showed percentages and embryo, and exalbuminous seed (Fig. 1 A-H). between 38% and 85%, and H. impetiginosus had The dimensions of the seed and embryo of the highest under these conditions (Table 2, Fig. Handroanthus impetiginosus were significantly 4 D-F). larger than those measured in H. ochraceus and The species studied have epigeal germination H. lapacho, except for the width of the seed body (Fig. 4 A-E). The morphology of the seedlings (Table 1, Fig. 1 A-F). Intermediate values of the obtained in the different trials was similar: stem seeds were recorded for H. ochraceus, although with short internodes, cotyledons with a central they showed few significant differences with notch, epicotyls with simple and trifoliate leaves those determined for H. lapacho (Table 1, Fig. 1 (protophylls or transitional leaves) and a curved C-F). Only H. ochraceus has polyembryonic seed. root with little branching (Fig. 4 E-H). Handroanthus impetiginosus showed the The factorial analysis showed that “species” greatest germination energy during the 12 is the principal factor affected, significantly all months of storage (Fig. 2). H. lapacho and the parameters evaluated (p< 0.001), whereas H. impetiginosus showed germination energy the “culture method” did not affect the epicotyl similar to that at the time of collection (33%). length and the germination percentage. Moreover, However, as from the fourth month of storage, the “species x culture methods” interaction was H. lapacho showed an abrupt decrease in this significant (p< 0.01) for the hypocotyl length, parameter, whereas H. impetiginosus increased its cotyledon width and the germination percentage values (Fig. 3). At eight months after storage the (Table 3). germination energy of H. ochraceus was lower in When analyzing the significant differences comparison with the other species studied (Fig. 2). (p< 0.05) in the epicotyl length it was seen that Similar germination percentages for H. H. impetiginosus showed the highest values, impetiginosus, H. lapacho and H. ochraceus (85% followed by H. ochraceus and then H. lapacho, to 98%) were seen at the time of collection (Fig. in agreement with findings in the parameters of 3). However, seeds of H. ochraceus gradually the seed and embryo previously described (Tables decreased their germination capacity to 63% 1, 2). after 4 months, reaching 43% at 12 months after Even though the differences found in the harvest (Fig. 3). The results for H. lapacho were hypocotyl length and cotyledon width are due similar but there was an abrupt decrease in the to the effect of the two independent factors and germination capacity at eight months after harvest their interaction (Tables 2, 3), it was seen that the (47%) (Fig. 3). The seeds of the species studied hypocotyl length was greater in the seedlings in that were stored at 5ºC for one year, mainly H. ex vitro culture; however this difference was not impetiginosus, showed germination percentages statistically significant inH. lapacho (Table 2; Fig. of 82-100% (Table 2; Fig. 3). 4 E-H). The cotyledon width of the seedlings of H. No significant differences were seen between ochraceus germinated in vitro showed significant the ex vitro and in vitro methods of germination differences (p< 0.05) in respect to the cotyledons in H. impetiginosus and H. ochraceus, but of the other species (Table 2). germination of H. lapacho was better in the in The dimensions of the seeds and embryos of vitro culture (Table 2). the three species studied exhibited a relationship Handroanthus impetiginosus, H. lapacho directly proportional to the size of the seedling. and H. ochraceus showed appropriate in vitro The larger seeds produce more vigorous seedlings, development in WPM (Table 2, Fig. 4 A-C). such as in H. impetiginosus (Tables 1, 2; Fig. The percentages of in vitro germination varied 1 A-F; 4 A-F). Furthermore, this species is the according to the species (49% to 82%) and the one that showed the greatest germination energy highest values were obtained in H. impetiginosus and percentage compared with the other species (Table 2, Fig. 4 D, E, G). The WPM supplemented studied.

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Fig. 2. Germination energy of Handroanthus impetiginosus, H. lapacho and H. ochraceus. Seeds cultivated in Petri dishes at the time of collection in the first season.

Fig. 3. Germination capacity of Handroanthus impetiginosus, H. lapacho and H. ochraceus. Seeds cultivated in Petri dishes at the time of collection in the first season.

Discussion Carvahlo et al., 2008; Nery et al., 2008). Sakai (2010) reports that most recalcitrant Several species of Handroanthus have species are associated with Dicotyledons germination problems in natural habitats. primitive or ancestral characters, such as large The process is extremely variable and exalbuminous seeds and a tropical woody differs between species and with the site habit. and conditions of seed formation (climate, Some authors show that in the mature seed phytosanitary status and physiological aspects) of representatives of the Bignoniaceae it is (Justiniano et al., 2000; Bocchese et al., 2008; common to see that the membranous cover, 10 N. M. Apóstolo et al., In vitro an ex vitro germination of Handroanthus

Table 1. Morphometric characteristics of the seeds of Handroanthus impetiginosus. H. lapacho and H. ochraceus from northwest Argentina (province of Salta). Different letters indicate significant differences between species at P> 0.05 (Tukey).

Seed Wings Seed body Embryo Species Length Width Thickness Width Length Width Length Width Thickness Midsection (mm) (mm) (mm) (mm) (mm) (mm) (mm) (mm) (mm) (mm) Handroanthus 5.5+0.5c 23.2+2.1c 0.6+0.1b 7.0+0.9b 4.8+0.4c 8.8 +1.0a 4.4+0.2c 6.6+0.2c 0.5+0.1b 2.6+0.1c lapacho

Handroanthus 7.6+0.9b 27.4+2.5b 0.6+0.1b 7.6+1.3b 7.0+1.0b 9.9+4.5a 6.1+0.3b 8.8+0.4b 0.4+0.1b 3.4+0.4b ochraceus Handroanthus 10.6+0.7a 37.6+2.9a 1.8+0.3a 13.0+1.6a 9.2+0.8a 11.8+1.0a 8.4+0.6a 10.3+0.8a 1.1+0.1a 4.5+0.5a impetiginosus

Table 2. Germination percentage and morphometric characteristics of the seedlings of Handroanthus impetiginosus. H. lapacho and H. ochraceus from in vitro and ex vitro cultivated seed. Seed of the second season grown after being preserved for a year at 5ºC. In vitro medium: WPM with 5 mm BA and activated carbon (5 g.L-1). Ex vitro substrate: soil:peat:perlite (1:1:1). Different letters indicate significant differences between species and treatments (P> 0.05).

Epicotyl Hypocotyl Cotyledon Germination Total Length Total Length Width (%) (mm) (mm) (mm) Handroanthus in vitro 3.4±1.4 c 12.6±1.7 b 9.8±2.7 c 57.8 b lapacho ex vitro 2.7±1.2 c 14.5±1.7 b 13.9±0.2 ab 38.1 c Handroanthus in vitro 9.1±1.9 b 13.8±1.5 b 17.8± 2.7 a 49.1 bc ochraceus ex vitro 8.5±1.0 b 41.4±1.1 a 16.3±0.4 a 58.7 b Handroanthus in vitro 41.1±8.0 a 15.1± 1.4 b 12.1±3.3 bc 81.7 a impetiginosus ex vitro 42.5±6.5 a 31.7±1.05 a 14.5±0.6 ab 85.0 a

Table 3. Ex vitro and in vitro germination. Analysis of variance for the effect of the factors (species and culture method) and their interaction with the morpho-physiological parameters of seedlings of Handroanthus impetiginosus. H. lapacho and H. ochraceus (P< 0.01).

Epicotyl Hypocotyl Cotyledon Germination Total Length Total Length Width % (mm) (mm) (mm) Gl F Sig. F Sig. F Sig. F Sig.

Species (S)* 2 176.10 0.000 6.87 0.001 23.83 0.000 99.67 0.000

Methods (M)** 1 0.43 0.535 17.76 0.000 7.04 0.009 0.02 0.892

S x M 2 1.28 0.280 17.14 0.000 6.80 0.000 12.27 0.00

Referencia: GI: Grados de libertad; F: Estadístico F de Fisher; Sig.: Significancia para P< 0.05. * Handroanthus impetiginosus. H. ochraceus or H. lapacho ** ex vitro or in vitro 11 BONPLANDIA 25(1). 2016

Fig. 4. Seedlings from ex vitro and in vitro germination. A-C: Iniciation of in vitro germination: A: Handroanthus ochraceus. B: H. impetiginosus. C: H. lapacho. D-E: Ex vitro germination of H. impetiginosus: D: Emergence of cotyledons. E: Developed seedling. F: Ex vitro seedling of H. ochraceus. G: In vitro seedling of H. impetiginosus after 30 days culture. H: Detail of the hypocotyl in seedling of H. ochraceus in in vitro culture. Bars: A-E, G: 2 cm; F, H: 1 cm.

composed of endothelium and the remains of 2008). Studies on seed ontogeny in Tabebuia the endosperm, is attached to the surface of the rosea, T. impetiginosa (= H. impetiginosus), embryo in a unique cordiform structure (Souza T. chrysotricha (= H. chrysotrichus), T. et al., 2005; Sampaio et al., 2007; Souza et al., serratifolia (= H. serratifolius), T. ochracea

12 N. M. Apóstolo et al., In vitro an ex vitro germination of Handroanthus

(= H. ochraceus) and T. pulcherima (= H. of the seed. Different authors have proposed pulcherrimus) have indicated that the seeds desinfection processes of seed of T. roseo- are considered exalbuminous because the alba, T. impetiginosa and T. serratifolia by cotyledons absorb most of the endosperm using (NaOCl) in concentrations of 2 to 10% through the endothelium before maturity. This active chlorine. However, they indicate that explains why embryos in seed of Tabebuia and high concentrations of this disinfectant reduce Handroanthus increase in size considerably the germination percentage (Carvahlo et al., (Silva et al., 2005; Sampaio et al., 2007; Sakai, 2008; Nery et al., 2008). The use of NaOCl in 2010). the germination trials of the three species of Moreover, von Teichman & Wyk (1994) Handroanthus ensured adequate disinfection indicate that the large embryo in exalbuminous of the seed and significant germination seed is an advantage for germination. The percentages. larger dimensions of the seed and embryo in H. Metabolism is slower under low temperature impetiginosus coincide with high germination so conservation under those conditions results percentages over the 12 months of trials. in a better germination capacity and lengthens According to Quinto et al. (2009), seed the life of the seed (Doria, 2010). Seed of dormancy in the seed of Handroanthus species, Handroanthus, mainly H. impetiginosus, mainly in cultivated H. impetiginosus, allows a conserved at 5ºC for one year showed high germination capacity over time. germination percentages up to 82-100% in both The presence of germination inhibitors seasons of collection. might be the cause of the gradual decrease No differences were seen between the in in germination capacity over a long time. vitro and ex vitro germination treatments, T. serratifolia showed a lower germination except in H. lapacho. Nery et al. (2008) percentage during the time when the seed had carried out studies comparing the in vitro and the highest content of polyphenols, since the ex vitro germination of T. serratifolia and oxidation products of these compounds affect they observed that the in vitro cultures gave germination as they reduce oxygen availability higher germination percentages. On the other in the seed (Carvahlo et al., 2008). hand, Silva et al. (2005) determined that the Moreover, H. ochraceus has polyembrionic optimum temperature for the germination of seed, as does H. chrysotrichus, due to the T. impetiginosa is 25ºC to 30ºC, coincident formation of one to four adventitious embryos with the range used in the in vitro and ex vitro by apomixis (Costa et al., 2004; Souza et germination trials of the three Handroanthus al., 2005; Sampaio et al., 2007). Apomitic species studied. species have a great ability to colonize habitats, The use of WPM for in vitro germination of leading to wide distribution compared with H. impetiginosus, H. ochraceus and H. lapacho non-apomitic species (Goldenberg & Shepherd, was appropriate for seedling development. The 1998). The dimensions observed in seed of solution of macronutrients alternative known H. ochraceus, together with the presence of as WPM (Lloyd & McCown, 1980) has been polyembryony, ensure the continuation of the formulated for woody species and has a 25% species in natural and exotic habitats. concentration of nitrate and ammonium ions According to Carvahlo et al. (2008) the seeds and a higher concentration of potassium and of T. serratifolia are mature enough to germinate sulphate than the Murashige-Skoog (MS) 53 days after anthesis which coincides with the formulation that is often used. Nery et al. dehiscence of the mature fruit for dispersal. (2008) showed that T. serratifolia had a higher Therefore, the seeds used in this study were germination percentage when the seeds were mature when collected, as the showed cultivated in media with the WPM formulation. clear evidence of the opening of the pericarp, However, Carvahlo et al. (2008) did not find enabling the analysis of germination energy and any significant differences between WPM and capacity. MS when cultivating embryos of the same One of the most serious problems in species. germination studies is fungal contamination The use of a substrate rich in organic matter

13 BONPLANDIA 25(1). 2016 for ex vitro trials ensured good germination preservation of native specimens. Finally, the percentages, especially in H. impetiginosus. In results for H. impetiginosus demonstrated its the ex vitro germination of T. heptaphylla (= H. capacity to perpetuate itself whether cultivated hepatphyllus), the clay substrate with additional or as a native tree. organic matter gave a greater quantity of germinated seed (42%), the radicles starting to appear between 6 to 10 days after the start of the Bibliography culture (Borchesse et al., 2008). Nevertheless, Fowler et al. (1998) found a higher germination BLAKESLEY, D., N. PASK, G. G. HENSHAW & M. percentage of T. cassinoides in sandy substrates F. FAY. 1996. Biotechnology and conservation compared with clay soils. of forest genetic resources: in vitro strategies and cryopreservation. Plant Growth Regul. 20: 11-16. The species studied have epigeal germination BOCCHESE, R. A., A. K. M. OLIVEIRA, A. M. and the morphological characteristics of the MELOTTO, V. FERNANDES & V. A. LAURA. seedling are similar to those observed by other 2008. Efeito de diferentes tipos de solos na authors in representatives of the genus. Ferreira germinaçao de sementes de Tabebuia heptaphylla & Cunha (2000) reported that the seedling em Casa Telada. Cerne, Lavras 14: 62-67. of T. caraiba have sinuous roots with little CARVAHLO, M. L. M., M. C.NERY, L. M. OLIVEIRA, ramification, a glabrous cylindrical hypocotyl, H. W. K. HILHORST & R. M. GUIMARAES. 2008. Morphophysiological development of Tabebuia persistent opposite cotyledons with a central serratifolia Vahl Nich. Sci. Agric. (Piracicaba, notch, epicotyls with short pedicels simple, Braz.) 65: 643-651. rarely trifoliate. Lozano & Zapater (2008) CORNER, E. J. H. 1976. The seeds of Dycotiledons. showed that the seedlings of H. impetiginosus Cambrigde University Press. USA. had six unifoliate protophylls and two trifoliate COSTA, M. E., D. S. SAMPAIO, A. A. S. PAOLI & S. transitional leaves with serrated borders at 60 C. A. L. LEITE. 2004. Poliembrinia e aspectos da days after ex situ germination. embriogênese em Tabebuia ochracea (Chamiso) The length of the hypocotyl was greater Standley (Bignonicaceae). Rev. Bras. Bot. 27: 395- 406. in ex vitro cultivated , although this DORIA, J. 2010. Generalidades sobre las semillas: difference was only statistically significant su producción, conservación y almacenamiento. in H. impetiginosus and H. ochraceus. The Cultivos Tropicales 31: 74-85. elongation of the hypocotyls may be associated FAY, M. F. 1994. In what situations is in vitro culture with the process of photomorphogenesis appropriate to plant conservation? Biodivers. (Doria, 2010; Zhang et al., 2010). However, the Conserv. 3: 176-183. light intensity in both culture methodologies FERREIRA, R. A. & M. C. L. CUNHA. 2000. analyzed (in vitro and ex vitro) was the same. It Aspectos morfológicos de sementes, plântulas e is possible that the differences observed might desenvolvimento da muda de craibeira (Tabebuia caraiba (Mart.) Burt.)-Bignoniaceae e pereiro be related to the composition of the culture (Aspidosperna pyrifolium Mart.)-Apocynaceae. Rev. substrate. The in vitro substrate contains 2% Bras. Sementes 22: 134-143. sucrose and there is evidence that an increase FOWLER, J. A., G. R. CURCIO, M. F. G. RACHWALL in the levels of this substance suppresses & Y. KUNIYOSHI. 1998. Germinaçao e vigor hypocotyl elongation (Zhang et al., 2010). de sementes de caixeta Tabebuia cassinoides DC On account of the seed characteristics and provenientes de diferentes solos orgánicos. Colombo: rapid loss of germination capacity together Embrapa Floresta. Comunicado Técnico Nº 25. with a restricted distribution, H. lapacho is GOLDENBER, G. R. & G. SHEPHERD. 1998. Studies a species with considerable conservation on the reproductive biology of Melastomataceae in “cerrado” vegetation. Plant Syst. Evol. 211: 13-29. problems in its natural habitat. In vitro GROSE, S. O. & G. OLMSTEAD. 2007. Evolution of a germination is a useful tool that could solve charismatic neotropical : molecular phylogeny this. On the other hand, H. ochraceus has seed of Tabebuia s.l. Crescentieae and allied genera of a size intermediate between the other species (Bignoniaceae). Syst. Bot. 32: 650-659. studied and its germination capacity gradually GROSE, S. O. & G. OLMSTEAD. 2007b. Taxonomic decreased. However, it is a seed that shows revisions in the polyphyletic genus Tabebuia s.l. polyembryony, ensuring efficiency in the (Bignoniaceae). Syst. Bot. 32: 660-670.

14 N. M. Apóstolo et al., In vitro an ex vitro germination of Handroanthus

IUCN. 2004. IUCN Red List of Threatened Species. www. SOUZA, L. A., M. C. IWAZALD & I. S. MOSHETA. iucnredlist.org. Accessed 28 December 2013. 2005. Morphology of the pericarp and seed of JUSTINIANO, M. J., T. S. FREDERICKSEN & D. NASH. Tabebuia chrysotricha (Mart. ex DC) Standl. 2000. Ecología y silvicultura de especies menos (Bignoniaceae). Braz. Arch. Biol. Techn. 48: 407- conocidas - Tajibos o Lapachos (Tabebuia spp.) Santa 418. Cruz, Bolivia. Editora El País. Bolivia. SOUZA, L. A., S. OLIVEIRA OYAMA & J. C. LOZANO, E. C. & M. A. ZAPATER. 2008. Delimitación MUNERATTO. 2008. Morphology and anatomy y estatus de Handroanthus heptaphyllus y H. of the developing fruit of Macfadyena unguis-cati impetiginosus (Bignoniaceae,Tecomeae). Darwiniana (L.)A.H. Gentry, Bignoniaceae. Acta Bot. Venez. 46: 304-317. 31: 1-13. LLOYD, G. & B. MCCOWN. 1980. Commercially- THORPE, T. A., I. S. HARRY & P. P. KUMAR. 1991. feasible micropropagation of mountain laurel, Kalmia Application of micropropagation to forestry. En: latifolia, by use of shoot-tip culture. Proc. Int. Plant DEBERGH, P.C. & R. H. ZIMMERMANN (eds.), Prop. Soc. (USA) 30: 421-427. Micropropagation. Technology and Application, NERY, M. C., M. L. M. CARVAHLO, L. M. OLIVEIRA, pp. 311-336. Kluwer Academic Publisher, The F. C. NERY & D. G. SILVA. 2008ª. Germinaçao in Netherlands. vitro e ex vitro de embrioes/sementes de Tabebuia VON TEICHMAN, I. & A. E. VAN WYK. 1994. serratifolia (Vahl.) Nich. Cerne, Lavras 14: 1-8. Structural aspects and trends in the evolution of QUINTO, L., P. A. MARTINEZ-HERNÁNDEZ, L. recalcitrant in Dicotyledons. Seed Sci. Res. 4: 225- PIMENTEL-BRIBIESCA & D. A. RODRIGUEZ- 239. TREJO, D. A. 2009. Alternativas para mejorar la ZAPATER, M. A., L. M. CALIFANO, E. M. DEL germinación de semillas de tres árboles tropicales. CASTILLO, M. A. QUIROGA & E. C. LOZANO. Rev. Chapingo 15: 23-28. 2009. Las especies nativas y exóticas de Tabebuia SAKAI, S. 2010. Evolution of exalbuminous seed as a y Handroanthus (Tecomeae, Bignoniaceae) en result of competition between maternally derived and Argentina. Darwiniana 47: 185-220. paternally derived genes. Evol. Ecol. Res. 12: 855-871. ZHANG, Y., Z. LIU, L. WANG, S. ZHENG, J. XIE & SAMPAIO, S., M. E. COSTA & A. A. S. PAOLI. 2007. Y. BI. 2010. Sucrose-induced hypocotyl elongation Ontogenia da semente de Tabebuia ochracea (Cham.) of Arabidopsis seedlings in darkness depends on Standl. (Bignoniaceae). Rev. Bras. Bot. 30: 289-302. the presence of gibberellins. J. Plant Physiol. 167: SILVA, E. A., A. C. DAVIDE, J. M. ROCHA FARIA, D. 1130–1136. L. BANDEIRA DE MELO & G. BARBOSA DE ZULOAGA, F. O., O. MORRONE & M. J. BELGRANO. ABREU. 2004. Germination studies on Tabebuia 2011. Catálogo de las Plantas Vasculares del Cono impetiginosa Mart. Cerne, Lavras 10: 1-9. Sur. www.darwin.edu.ar. Accessed 23 January 2014.

Original recibido el 21 de marzo de 2016; aceptado el 26 de abril de 2016.

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